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PubMed1995

Separation of bacterial cells by free flow electrophoresis under microgravity: a result of the SpaceLab-Japan project on Space Shuttle flight STS-47.

We demonstrated free flow electrophoresis (FFE) of charged cells under microgravity, where gravitational effects are almost eliminated. Separation of a mixture of three bacterial strains (mutants of Salmonella typhimurium LT2) by FFE was conducted on NASA Space Shuttle flight STS-47 (September 1992). The experiment was designed to differentiate three strains having different lipopolysaccharide core structures in the cell membrane. The results were compared to those of ground experiments, in order to examine whether or not FFE in a weightless environment provides distinct advantages. Smooth strain SL1027 and rough strain SL3749 migrated to two separated fractions. The quality (viability) and the yields of the separated samples were sufficient to show the advantage of microgravity. Another rough strain, SL1102, exhibited unexpected electrophoretic behavior, which prevented the complete resolution of the three strains. All the strains were recovered as viable cells after 8 days of flight. The present study suggests that electrophoretic separation of bacterial cells is much more effective under microgravity conditions with relatively good resolution in comparison with the ground operation.

Anti-Bacterial Agents↗

PubMed1999

The effectiveness of two sterilization methods when different precleaning techniques are employed.

The effectiveness was investigated of methods for the preparation of dental handpieces prior to sterilization procedures utilizing ethylene oxide (ETO) gas. The handpieces were cleaned using either a forced-air purging unit (group 1) or by flushing with air and water from the dental unit (group 2). They were inoculated with either Bacillus subtilis or Streptococcus mutans. After exposure to either steam or ETO gas, the handpieces were flushed with saline and the viability of recovered bacteria assessed. No viable bacteria were recovered from group 1 handpieces treated with either ETO gas or steam. However, viable S. mutans were recovered from group 2 handpieces following exposure to ETO gas. Thus, the use of a high-pressure forced-air purging unit may be required for the reliable sterilization of dental handpieces by ETO gas, as viable S. mutans could be recovered from untreated handpieces exposed to ETO gas.

Air Pressure↗

PubMed2006

Concentration of Bacillus spores by using silica magnetic particles.

Silica particles are mainly used for the concentration of nucleic acid for diagnostic purposes. This is usually done under acidic or chaotropic conditions that will demolish most of the living organisms and prevent the application of other diagnostic tests. Here we describe the development of a method for the capturing and concentration of Bacillus spores using silica magnetic particles to enable fast and sensitive detection. We have shown that capturing various Bacilli spores via silica magnetic particles is limited, with large differences between spore batches (42 +/- 25%). The hydrophobic exosporium layer of spore limits the capture by the hydrophilic silica beads. Partial removal of Bacillus exosporium increases capture efficiency. To increase capturing efficiency without harming the spores' viability, a cationic lipid, didecyldimethylammonium bromide (DDAB), was used as a coat for the negatively charged silica particles. DDAB treatment increased capture efficiency from 42% to more than 90%. Using this method, we were able to capture as few as 100 Bacillus anthracis spores/mL with 90% efficacy. Release of captured spores was achieved by the addition of albumin. The capture and release processes were verified by plating and by flow cytometry using light scatter analysis. The method is simple, efficient, easy to operate, and fast.

Adsorption↗

PubMed2001

Co-option of endocytic functions of cellular caveolae by pathogens.

It is increasingly becoming clear that various immune cells are infected by the very pathogens that they are supposed to attack. Although many mechanisms for microbial entry exist, it appears that a common route of entry shared by certain bacteria, viruses and parasites involves cellular lipid-rich microdomains sometimes called caveolae. These cellular entities, which are characterized by their preferential accumulation of glycosylphosphatidylinositol (GPI)-anchored molecules, cholesterol and various glycolipids, and a distinct protein (caveolin), are present in many effector cells of the immune system including neutrophils, macrophages, mast cells and dendritic cells. These structures have an innate capacity to endocytoze various ligands and traffic them to different intracellular sites and sometimes, back to the extracellular cell surface. Because caveolae do not typically fuse with lysosomes, the ligands borne by caveolar vesicles are essentially intact, which is in marked contrast to ligands endocytozed via the classical endosome-lysosome pathway. A number of microbes or their exotoxins co-opt the unique features of caveolae to enter and traffic, without any apparent loss of viability and function, to different sites within immune and other host cells. In spite of their wide disparity in size and other structural attributes, we predict that a common feature among caveolae-utilizing pathogens and toxins is that their cognate receptor(s) are localized within plasmalemmal caveolae of the host cell.

Adhesins, Bacterial↗

PubMed1998

Antibacterial effects of a bioactive glass paste on oral microorganisms.

Bioactive glasses contain oxides of calcium, sodium, phosphorus, and silicon in a proportion that provides the material with surface activity and concomitantly with the property of forming a strong bond with bone. Bioactive glasses have been tested as bone substitutes in different clinical situations. In an aqueous environment, Ca2+, Na+, PO4(3-) , and Si4+ are released from the glass, resulting in a rise in pH and in osmotic pressure in its vicinity. Since these are factors that potentially influence the viability of oral microorganisms at the dentogingival margin, we studied the effects of bioactive glass S53P4 on the oral microorganisms Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, Actinomyces naeslundii, Streptococcus mutans, and Streptococcus sanguis. This was done by incubating each microbe in a suspension, in the presence of bioactive glass S53P4 in powder form. A. naeslundii was found to lose its viability within 10 min under the experimental conditions. A. actinomycetemcomitans, P. gingivalis, and S. mutans lost their viability within 60 min. Also for S. sanguis a significant loss of viability was seen within 60 min, but it was the only microbe that had any viable cells left after 60 min. Thus, in aqueous solutions the powdered bioactive glass S53P4 appears to have a broad antimicrobial effect on microorganisms of both supra- and subgingival plaque. Consequently, it could be useful as an ingredient in tooth-care products that may have beneficial effects on oral health both from a cariologic and a periodontal point of view.

Actinomyces↗

PubMed1994

Survival of Staphylococcus aureus in lakewater monitored by flow cytometry.

The ability of flow cytometry to detect and enumerate viable bacteria during survival in a lakewater microcosm was assessed using Staphylococcus aureus as a model organism. Counts of colony-forming units (c.f.u.) on nutrient agar were not significantly different from those obtained by flow cytometric detection of rhodamine 123 stained bacteria and there was no evidence for a viable but nonculturable state using these methods. However c.f.u. were significantly lower when estimated using mannitol salts agar compared with nutrient agar. S. aureus was also enumerated immunofluorescently after staining with FITC-IgG. There was no significant difference between the population estimated immunofluorescently and by acridine orange direct counting, and unlike estimations of viability, only slight reductions in total cell numbers were observed. Changes in the protein and nucleic acid content of S. aureus during survival were also measured by flow cytometry to investigate any potential heterogeneity arising within the starved population. Flow cytometric determinations were found to correlate significantly with their respective chemical determinations. These results demonstrate the ability of flow cytometry to detect viable bacteria during starvation and to study changes in macromolecular content. They also illustrate the importance of using appropriate methods for the detection of viable bacteria in environmental samples.

Bacterial Proteins↗

PubMed1980

Lethal effect of a heterologous murein hydrolase on penicillin-treated Streptococcus sanguis.

Nine strains of Streptococcus sanguis exhibited tolerance to benzylpenicillin: the growth of each strain was susceptible to penicillin with minimal inhibitory concentrations of 0.1 mug/ml or lower, but the bacteriolytic and bactericidal effects were limited in each case. The tolerance of these bacteria was also reflected in the large discrepancies between the minimal inhibitory and minimal bactericidal concentrations for benzylpenicillin. The hypothesis that a natural deficiency of endogenous murein hydrolase (autolysin) in this species accounts for the penicillin tolerance was tested by using a heterologous murein hydrolase, the C-phage-associated lysin. In seven of the strains, addition of the lysin to the culture together with penicillin or other cell wall inhibitors resulted in lysis and rapid loss of viability. The enzyme alone did not appreciably affect normally growing cultures. The irreversible effects of penicillin plus lysin were drastically reduced in the presence of the bacteriostatic agents chloramphenicol and cerulenin. Speculations based on experiments are presented for the mechanisms by which penicillin treatment sensitizes these bacteria to an exogenous lytic enzyme. Similar phenomena requiring cooperation of host factors and penicillin may occur during infection, since somewhat similar although less pronounced results were obtained by addition of human lysozyme to penicillin-treated S. sanguis.

Amidohydrolases↗

PubMed1970

T-even bacteriophage-tolerant mutants of Escherichia coli B. I. Isolation and preliminary characterization.

A general procedure is described for isolation of T-even phage-tolerant mutants of Escherichia coli. Two such mutants of E. coli B have been examined in some detail. These mutants adsorb T-even phages but are unable to release viable progeny. Under certain conditions, viability of the cells is completely unaffected by phage infection in one mutant, and there is but a slight decrease in colony-forming ability in the other. Phage deoxyribonucleic acid (DNA) is injected into these cells, as shown by the formation of phage-specific enzymes, but it is not degraded to acid-soluble material. Some phage DNA replication occurs in both strains. The mutants are both more resistant to ultraviolet light than is the parent strain.

Adsorption↗

PubMed1976

Hepatocyte perfusion within a centrifuge.

A centrifuge, as used in blood banks for washing erythrocytes, is shown to be a good perfusion chamber for hepatocyte suspensions being evaluated for potential extracorporeal liver support. The perfusate was cell-free oxygenated--pO2 300 millimeters of mercury--plasma, serum or tissue culture medium. A centrifuge that requires intermittent perfusion cannot deliver sufficient oxygen to maintain prolonged viability. A continuous flow unit can, at a flow of 800 milliliters per minute, support most functions of 100 grams of hepatocytes for six hours and some function for 12 hours. Metabolism of 2-14C tagged pyruvate to 14CO2 is the most useful sensitive measurement of liver function in this system. It is concluded that a centrifuge allowing a constant perfusion of oxygenated plasma into hepatocytes kept within the unit by centrifugal force is a suitable base for extracorporeal liver support.

Animals↗

PubMed1990

[The combined action of microwave radiation and hydrogen peroxide on the viability and ultrastructure of Pseudomonas aeruginosa cells].

The comparative study of the action of microwave radiation and hydrogen peroxide, as well as their combined action, on the viability and ultrastructure of P. aeruginosa cells has been made. The combined use of microwave radiation and hydrogen peroxide has been shown to decrease the viability of P. aeruginosa 1.5-2 times in comparison with the isolated action of each factor. The electron microscopic study of the ultrastructure of cells have shown the deterioration of the surface structures, nuclear and ribosomal apparatus of the cells under the isolated action of each of the above-mentioned factors or under their combined action. The morphological picture of these changes has proved to be different. The maximum changes in the ultrastructure of P. aeruginosa cells have been registered after the combined action of these bactericidal factors.

Colony Count, Microbial↗

PubMed2005

Assessment of a new technique combining a viability test, whole-cell hybridization and laser-scanning cytometry for the direct counting of viable Enterobacteriaceae cells in drinking water.

A new direct approach, called direct viable count (DVC)-FISH-ScanRDI, combining viability measurement, specific detection and sensitive enumeration of highly diluted Enterobacteriaceae cells, was assessed during the summer in water samples from a North American drinking water treatment plant and its distribution system. Major results of this field investigation show a higher sensitivity of the DVC-FISH-ScanRDI approach in enumerating viable Enterobacteriaceae cells in distributed drinking water, relative to a culture-based method, and the increased concentration of viable but non-culturable (VBNC) Enterobacteriaceae cells in distributed water for temperatures above 18 degrees C.

Bacteriological Techniques↗

PubMed1983

Methodological considerations in culturing peptidergic neurons.

Both explant and dispersed cell culture preparations of brain tissue provide a means to directly assess the functions of neuropeptide-containing brain cells isolated from the complex influences present in vivo. The validity of the approach depends on reproducibility of observations. In dispersed cell cultures, we find that cell responses, as determined by secretion of the peptide somatostatin, have remained relatively constant both quantitatively and qualitatively over numerous preparations. In addition, for pharmacologic studies on somatostatin secretion, data from several laboratories are in good agreement. The validity of the dispersed cell approach also depends on whether the pharmacologic and physiologic behavior of cells parallels that expected of excitable tissue. The variability of the explant cultures from culture dish to culture dish makes quantitative experiments, such as demonstrated with the dispersed cultures, difficult. On the other hand, explant cultures better maintain the integrity of the tissue components, so that interactions between neurons and glial cells could occur as in vivo. The long-term health and viability of neuropeptidergic cells in explant and dispersed culture make both preparations potentially useful models to examine central nervous system physiology. Future work with such preparations must eventually address the problems of culturing adult brain tissue, the precise nutrient and hormonal requirements of brain cells, so that undefined medium components, such as serum, can be eliminated from the culture environment, and the general question of whether observations made in vitro facilitate our understanding of intact brain physiology.

Animals↗

PubMed1986

Analogues of N-benzyloxydihydrotriazines: in vitro antimalarial activity against Plasmodium falciparum.

The antimalarial activities of a series of chlorophenyloxyalkoxy and chlorophenalkoxy N-substituted diamino-dihydrotriazines were determined in vitro against three strains of Plasmodium falciparum (Malayan Camp, Vietnam Smith, FCB) with diverse levels of resistance to chloroquine, pyrimethamine, and cycloguanil. Parasite viability was assayed by the inhibition of the uptake of radiolabelled hypoxanthine. Most of the ID-50S of these compounds were less than 1.0 ng ml-1. Consistent differences in sensitivities to these compounds were observed and appeared to be strain related. The Malayan Camp was the most sensitive and Vietnam Smith was the least sensitive. These differences appeared to be related primarily to an inherent sensitivity of a particular strain to the series of analogues examined rather than to a pattern of cross-resistance to chloroquine, pyrimethamine, or cycloguanil.

Antimalarials↗

PubMed2005

Effect of electrophoretically activated calcium hydroxide on bacterial viability in dentinal tubules--in vitro.

To evaluate the ability of electrophoretically activated calcium hydroxide (CH) to eliminate bacteria in dentinal tubules. In an in vitro model of dentinal tubule infection, 18 cylindrical root specimens prepared from freshly extracted bovine teeth were used. After removal of the smear layer, intracanal dentinal tubules were infected with Enterococcus faecalis for 21 days. In 12 specimens, CH paste was placed in the root canals for 7 days. In six of these, an electrophoretic current (10 mA per 10 min), using two electrodes, was applied after placing the medicament in the canal. Powder dentin samples obtained from within the canal lumina using ISO 025, 027, 029, 031 and 033 burs were examined for the presence of vital bacteria by inoculating agar plates and counting colony forming units. anova with repeated measures was used to analyze results. A significant difference was found between experimental groups and the positive control group. CH and electrophoretically activated CH significantly (P <0.001) reduced bacterial viabilities in dentinal tubules to a depth of 200 microm. Treatment with electrophoresis was significantly (P <0.001) more effective than pure CH in depths of 200-500 microm. Specimens treated with electrophoretically activated CH showed no viable bacteria in dentinal tubules to a depth of 500 microm from the root canal space within 7 days. The time required for treatment of pulpal infection root resorption may be decreased, thus minimizing the risk of coronal fractures in young patients with traumatized teeth.

Analysis of Variance↗

PubMed1999

Further characterization of human salivary anticandidal activities in a human immunodeficiency virus-positive cohort by use of microassays.

Salivary anticandidal activities play an important role in oral candidal infection. R. P. Santarpia et al. (Oral Microbiol. Immunol. 7:38-43, 1992) developed in vitro anticandidal assays to measure the ability of saliva to inhibit the viability of Candida albicans blastoconidia and the formation of germ tubes by C. albicans. In this report, we describe modifications of these assays for use with small volumes of saliva (50 to 100 microl). For healthy subjects, there is strong inhibition of blastoconidial viability in stimulated parotid (75%), submandibular-sublingual (74%), and whole (97%) saliva, as well as strong inhibition of germ tube formation (>80%) for all three saliva types. The susceptibility of several Candida isolates to inhibition of viability by saliva collected from healthy subjects is independent of body source of Candida isolation (blood, oral cavity, or vagina) or the susceptibility of the isolate to the antifungal drug fluconazole. Salivary anticandidal activities in human immunodeficiency virus (HIV)-infected patients were significantly lower than those in healthy controls for inhibition of blastoconidial viability (P < 0.05) and germ tube formation (P < 0. 001). Stimulated whole-saliva flow rates were also significantly lower (P < 0.05) for HIV-infected patients. These results show that saliva of healthy individuals has anticandidal activity and that this activity is reduced in the saliva of HIV-infected patients. These findings may help explain the greater incidence of oral candidal infections for individuals with AIDS.

AIDS-Related Opportunistic Infections↗

PubMed1992

Adherence and entry of Borrelia burgdorferi in Vero cells.

Adherence to and entry of the parasite into the host is one of the essential elements of microbial pathogenicity. We investigated the adherence to and entry into primate kidney epithelial (Vero) cells of Borrelia burgdorferi by radiolabelling techniques, immunofluorescence and electronmicroscopy. The attachment to and subsequent entry of both untreated and heat (50 degrees C)-treated B. burgdorferi into Vero cells occurred at cell-surface sites associated with aggregated coated pits. In contrast, there was minimal attachment of spirochaetes heated at 60 degrees C. Radiometric studies showed that, with untreated cells, there was incorporation of both 14C-glucose-1-phosphate and 14C-thymidine, whereas with the 50 degrees C-treated spirochaetes only glucose-1-phosphate was incorporated, and with the 60 degrees C-treated spirochates neither radionuclide was incorporated. Spirochaetes heated at 50 degrees C or 60 degrees C did not grow at 35 degrees C in culture medium. These results suggest that the presence of certain metabolic activities of the spirochaete but not viability (ability to grow) are necessary for the attachment process. After entry of untreated B. burgdorferi, most of the spirochaetes were either free in the cytoplasm or tightly bound to the host membrane. In contrast, 50 degrees C-treated spirochaetes remained bound to host membrane in large phagosome-like vesicles.

Animals↗

PubMed1970

The action of streptomycin in a mutant of Escherichia coli with increased sensitivity to the antibiotic.

A mutant of Escherichia coli with increased sensitivity to streptomycin has been studied. This strain differed from a normal str(s) strain in that streptomycin produced inhibition of protein synthesis and loss of viability with almost no lag period. Chloramphenicol protected a normal str(s) strain but not the mutant against the bactericidal action of streptomycin. The results obtained support the idea that access of streptomycin to its site of action in a normal cell is restricted, and that this restriction, which is much less effective in the mutant, probably involves a permeability barrier. Comparison of the inhibition of protein synthesis by streptomycin with concomitant changes in the distribution of polyribosomes in both strains suggested that the antibiotic can directly inhibit the translation of mRNA.

Bacterial Proteins↗

PubMed1993

The pH as an important determinant of sperm-mucus interaction.

OBJECTIVE: To determine the clinical significance of endocervical mucus pH on sperm-mucus interaction during infertility investigation. PATIENTS AND MATERIAL: Two hundred sixteen couples with a median duration of infertility of 4 years (range, 1 to 19 years) presenting at the infertility unit of the Women's University Hospital of Heidelberg, Germany. MAIN OUTCOME MEASURES: Determination of endocervical pH by colorimetric and electrometric measurement and correlation of results with the outcome of postcoital testing (PCT) and other parameters of infertility investigation (semen and cervical mucus [CM] quality, microbial colonization of cervix and ejaculates, medical history, hormonal status, and specific medication) and the subsequent fertility in a prospective study. In vitro experiments with the sperm-cervical mucus penetration test (SCMPT) used as biological model. RESULTS: The colorimetric determination of endocervical mucus pH is an easy method, suitable for routine clinical use, correlating significantly with electrometric measurement of pH. Median pH was 7.0 (range, 5.4 to 8.2). The mucus pH was significantly related with the results of PCT, even when mucus and semen variables were taken into account. No significant relationship was seen between the cervical index and mucus pH and the microbial colonization of cervix and ejaculates. The pH of endocervical secretions correlated with the peripheral hormonal status: low pH levels were significantly more frequent in patients with hyperandrogenemia, indicated by high testosterone and/or dehydroepiandrosterone sulfate levels before medication was started, and in hyperandrogenemic patients treated with dexamethasone than in the other women. Oral administration of estrogens led to a subtle alkalinization of the CM. With regard to subsequent fertility 6 months after pH testing, the pregnancy rate was significantly lower in women offering reduced mucus pH on occasion of the PCT in the group of couples with primary infertility and in couples with oligozoospermia of the male partner. The significant influence of pH on sperm-mucus interaction was confirmed in vitro with the SCMPT. CONCLUSIONS: The results indicate that the pH of the CM, easily determined with pH indicator paper, is an important parameter of mucus quality with significant influence on spermatozoal viability in CM, which correlates with peripheral hormonal status and can be affected by oral medication with estrogens. Therefore the routine determination of pH on occasion of the PCT is recommended during infertility investigation.

Adult↗